The active transport of small molecules across biological membranes is a fundamental property of all cells. Transport proteins must (i) specifically recognize substrates, (ii) move them from one side of the membrane to the other and (iii) couple a source of energy to substrate movement. The long term objective of the research described in this proposal is to understand these steps and their regulation at the molecular level. To do this a molecular genetic analysis of the maltose transport system of Escherichia coli will be carried out. This system is composed of a periplasmic maltose binding-protein (MBP), and three membrane proteins MalF,G,K. MBP is required for transport and interacts with the MalF and MalG proteins. The MalF and MalG proteins also contain a gated substrate recognition site. The MalK protein has a nucleotide binding fold and shares extensive sequence similarity with many ATP binding proteins involved in diverse biological functions. Some of these related proteins include the mdr P-glycoproteins that are involved in tumor cell multiple drug resistance. Mutations which perturb the interaction of MBP with MalF and MalG will be isolated and sequenced. Mutations in the malF and malG genes that affect the accessibility of the substrate recognition site will be localized by DNA sequencing. This information will determine which regions form the gate that controls access to the substrate recognition site. The role of ATP binding at the nucleotide binding fold of the MalK protein will be evaluated. Transport defective malK mutants in which ATP binding no longer occurs will be studied. Revertants of these mutants that regain transport activity will be isolated and characterized. Attempts to replace the MalK function with other proteins of similar structure will be made. Genes which encode the other proteins that can replace MalK will be identified. Mutations in the malK gene that affect the regulatory functions of MalK will be isolated. These include mutations that abolish the ability of MalK to metabolize endogenous inducers of the mal regulon and mutations that make the mal system resistant to inhibition by the glucose-specific EIII of the PTS system. It is anticipated that these experiments will result in detailed knowledge of the important